Novel Pheretima guillelmi-derived antithrombotic protein DPf3: Identification, characterization, in vitro evaluation and antithrombotic mechanisms investigation

Wu, Yang‐Chang; Hu, Shaonan; Ma, Yunnan; Zhao, Bo; Yang, Wanqing; Lu, Yang; Li, Pengyue; Du, Shouying

doi:10.1016/j.ijbiomac.2020.03.097

Cited by 21 publications

(12 citation statements)

References 37 publications

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“…At present, some thrombolytic drugs, such as recombinant tissue-plasminogen activator (rt-PA), urokinase (uPA), and streptokinase (SK), have been widely used to treat thrombosis disorders. However, their side effects, such as heavy hemorrhagic complications, allergic reactions, anaphylaxis, and immunoreactions, are often reported, low specificity for fibrin and relatively high cost are also their deficiencies in clinical application ( Vernooij et al, 2009 ; Wu et al, 2020 ). As a vicarious therapy, traditional medicinal animals such as earthworms, snakes, and leeches have attracted more attention in the past few decades ( Wu et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation

Liu

Yang

et al. 2022

Front. Mol. Biosci.

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Thrombotic diseases have been considered major causes of death around the world. Treatments with thrombolytic drugs, such as recombinant tissue-plasminogen activator, urokinase, and streptokinase, are reported to have a life-threatening bleeding tendency. On the contrary, lumbrokinase, identified from Lumbricus rubellus, is specific to fibrin and does not cause excessive bleeding. It possesses fibrinolytic activity and activation of plasminogen to dissolve fibrin. Hence, the purification of fibrinolytic protein monomer from earthworm and antithrombotic evaluation and investigation of mechanisms are needed. In this study, a novel fibrinolytic protein EPF3, with strong fibrinolytic activity, was purified from Pheretima vulgaris by ion exchange and size exclusion chromatography. SDS PAGE, bottom-up proteomics analysis, de novo sequencing, and circular dichroism (CD) analysis were carried out for identification and characterization of it. EPF3, with a molecular weight of 25136.24 Da, consisted of 241 amino acids and contained various forms of secondary structures, including α-helix (3.9%), β-sheet (42.8%), β-turn (21.2%), and random coil (32.1%). It was a trypsin-like serine protease and stable at pH 7.0 to 11.0 and below 40°C. EPF3 was confirmed to possess an antithrombotic effect by ex vivo clot lysis test and fibrinogen-thrombin time (Fib-TT) assay. The three-dimensional structure of EPF3 was predicted by SWISS-MODEL. Molecular docking analysis predicted that EPF3 could directly interact with antithrombotic target proteins (fibrin, fibrinogen, and plasminogen), which was further confirmed by further studies. The antithrombotic mechanism of EPF3 was clarified to be outstanding direct fibrinolysis, fibrinogenolytic activity, and certain activation of plasminogen. EPF3 possesses the potential to be developed into a promising antithrombotic agent.

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Section: Introductionmentioning

confidence: 99%

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation

Liu

Yang

et al. 2022

Front. Mol. Biosci.

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“… 51 There is a research revealed that Pheretima may have anti-coagulant and anti-thrombotic functions through regulating DPf3, which is a group of antithrombotic functional proteins purified from Pheretima . 52 Moreover, Bombyx Batryticatus has been reported to exhibit pharmacological activity on the nervous system, such as anticonvulsant and antiepileptic effects, hypnotic effects, and neurotrophic effects and so on. 17 It has been also reported that (4E,6E,2S,3R)-2-N-docosanoyl-4,6-tetradecasphingadienine is the potential structure in Bombyx Batryticatus with a neurite outgrowth promoting activity.…”

Section: Discussionmentioning

confidence: 99%

An Integrated Analysis of Network Pharmacology and Experimental Validation to Reveal the Mechanism of Chinese Medicine Formula Naotaifang in Treating Cerebral Ischemia-Reperfusion Injury

Yang¹,

Chen²,

Mei³

et al. 2021

DDDT

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Background: Cerebral ischemia-reperfusion injury (CIRI) is a crucial factor leading to a poor prognosis for ischemic stroke patients. As a novel Chinese medicine formula, Naotaifang (NTF) was proven to exhibit a neuroprotective effect against ischemic stroke, clinically, and to alleviate CIRI in animals. However, the mechanisms underlying the beneficial effect have not been fully elucidated. Methods: In this study, we combined a network pharmacology approach and an in vivo experiment to explore the specific effects and underlying mechanisms of NTF in the treatment of ischemia-reperfusion injury. A research strategy based on network pharmacology, combining target prediction, network construction, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and molecular docking was used to predict the targets of NTF in treating the ischemic stroke and CIRI. On the other hand, we used HPLC and HRMS to identify biologically active components of NTF. Middle cerebral artery occlusion models in rats were utilized to evaluate the effect and the underlying mechanisms of NTF against CIRI after ischemic stroke. Results: Network pharmacology analysis revealed 43 potential targets and 14 signaling pathways for the treatment of NTF against CIRI after ischemic stroke. Functional enrichment analysis showed that a STAT3/PI3K/AKT signaling pathway serves as the target for in vivo experimental study validation. The results of animal experiments showed that NTF significantly alleviated CIRI by decreasing neurological score, infarct volume, numbers of apoptotic neuronal cells, increasing density of dendritic spines and survival of neurons. Furthermore, NTF could increase the expression of p-STAT3, PI3K, p-AKT. In addition, the detection of apoptosis-related factors showed that the NTF could raise the expression of Bcl-2 and reduce the expression of Bax. Conclusion:This network pharmacological and experimental study indicated that NTF, as a therapeutic candidate for the management of CIRI following ischemic stroke, may exert a protective effect through the STAT3/PI3K/AKT signaling pathway.

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“…Although many fibrinolytic agents have been developed and used for clinical purposes, thromboembolic diseases remain the leading cause of adult morbidity and mortality in the world [ 3 ]. Increasing attention has been applied in recent years to the development of effective agents for clinical application from various unusual animal species [ 2 ].…”

Section: Discussionmentioning

confidence: 99%

“…As commonly known, thrombosis is a common underlying factor for the occurrence of cerebro-cardiovascular diseases, such as stroke, heart attacks, and pulmonary embolisms [ 1 , 2 ]. Thrombotic diseases present a significant risk of mortality and disability, which has seriously affected public health [ 3 ]. Dissolving the thrombus is essential in the treatment of thrombotic diseases.…”

Section: Introductionmentioning

confidence: 99%

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases

Yang

Wang

et al. 2021

Molecules

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Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression.

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Novel Pheretima guillelmi-derived antithrombotic protein DPf3: Identification, characterization, in vitro evaluation and antithrombotic mechanisms investigation

Cited by 21 publications

References 37 publications

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation

An Integrated Analysis of Network Pharmacology and Experimental Validation to Reveal the Mechanism of Chinese Medicine Formula Naotaifang in Treating Cerebral Ischemia-Reperfusion Injury

Bioevaluation of Pheretima vulgaris Antithrombotic Extract, PvQ, and Isolation, Identification of Six Novel PvQ-Derived Fibrinolytic Proteases

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